PROJECT SUMMARY/ABSTRACT Type 2 diabetes places a significant and growing health burden on the world, with 440 million cases predicted by the year 2030 worldwide. In type 2 diabetes, increasing insulin resistance demands greater and greater insulin production from pancreatic beta cells. The resulting stress on the protein folding machinery of the beta cells triggers the unfolded protein response (UPR), a cell signaling network that acts to restore homeostasis in the face of cell stress. However, chronic UPR that fails to restore cell homeostasis can instead drive cell death. The result is a feed-forward loop with increased stress on the remaining pancreatic beta cells and eventual loss of systemic glucose homeostasis, leading to devastating end-organ damage in the kidneys, eyes, heart, and peripheral nervous system. The current project proposal aims to improve our ability to visualize and characterize the gene networks underlying UPR regulation and the switch between pro-survival and pro-cell death pathways. UPR activation starts with three molecular sensors of stress: IRE1?, PERK, and ATF6. Under chronic UPR, the transcription factor CHOP is upregulated, and it activates downstream pathways that lead to cell death. Aim 1 of the proposed project characterize the dynamics of UPR pathway activation using new cell lines with fluorescent reporters for the UPR components IRE1?, PERK, ATF6, and CHOP. These reporters will enable the observation of switches in UPR states in at the level of individual cells. This system should offer a new tool to the fields of cell stress and type 2 diabetes, and it could also be used in the future to screen for pharmaceutical agents that affect specific arms of the UPR pathway. A key challenge to understanding and treating diseases like type 2 diabetes is their complexity, with crosstalk among multiple cell pathways. Aim 2 will identify combinations of genes that modulate the UPR in pancreatic beta cells and that protect cells from the toxic effects of cell stress, using a novel, high-throughput approach recently developed for investigating the phenotypic effects of gene combinations (CombiGEM). This combinatorial genetic approach may better model UPR regulation and its interactions with other key regulatory cell pathways. Candidate interactions identified by CombiGEM in immortalized beta cell lines will be validated in primary murine beta cells. Ultimately, these studies may offer insight into the modulation of the UPR in type 2 diabetes, with potential therapeutic applications.